(1) Field of the Invention
The present invention relates to novel oxalic acid ester derivatives useful for quantitatively measuring a small amount of fluorescent substances, and hydrogen peroxide itself or produced, for instance, in enzyme immuno assay.
(2) Description of the Prior Art
An active intermediate (1,2-dioxethanedione) which is formed by an oxalic acid ester and hydrogen peroxide excite the fluorescent substances and make emission thereof, although there may be difference in quantum yield, so long as the substances have fluorescence from the visible range to the infrared range. The reaction systems thereof is used for quantitative measurement of hydrogen peroxide, substances participating in enzymatic reactions to produce hydrogen peroxide, and the like. Further, the fluorescent substances as receptors for energy from the active intermediates can also be measured thereby. Moreover, these quantitative measurements have been adopted as a methods of quantitatively measuring with high sensitivity a very small amount of hydrogen peroxide produced, for instance, in enzyme immuno assay and a method of quantitatively measuring with high sensitivity a fluorescent substance through being subjected to a main reaction after separated by high performance liquid chromatography (HPLC). ##STR3##
It is indispensable to obtain a rellable measured value by using a very small amount of a sample in the field in which diagnosis or treatment is performed through grasping the intracorporeal dynamics of a component of a living body, and the highly sensitive quantitatively measuring methods using the above reaction have been successively developed. For instance, there have been disclosed a quantitative measurement of hydrogen peroxide in the enzyme immunoassay of 17 .alpha.-hydroxyprogesterone [Arakawa et at., Chem. Pharm. Bull., 30, 3036, (1982)] and so on, quantitative measurements of urinary glucose [Williams et al., Anal. Chem., 48, 1003(1976)] and the activity of lactate dehydrogenase [Williams et al., Anal. Chem., 48, 1478(1976)], a quantitative measurement of dansylamino acid in HPLC [Kobayashi et al., Anal. Chem., 52, 424(1980)], a quantitative measurement of fluoresamine labeled cathecoalamine [Kobayashi et al., Anal. Biochem., 112, 99(1981)] and so on.
Among the chemiluminescent reagents giving the above high sensitive chemiluminescence, there are many compounds offered as the oxalic acid esters. For instance, use may be ordinarily made of bis(2,4,6-trichlorophenyl)-oxalate(hereinafter abbreviated as TCPO), bis(2,4-dinitrophenyl)oxalate(hereinafter abbreviated as DNPO), bis(pentachlorophenyl)oxalate, bis(4-nitro-3-trifluoromethyl)oxalate, bis(4-nitro-2-formylphenyl)-oxalate, bis(pentafluorophenyl)oxalate and so on.
However, there is no limit for needs in the scientific world, and the conventional chemiluminescent reagents have not been satisfactory in light of the requirements that sensitivity is higher; even when the oxalic acid esters is used at a high concentration, high emission intensity can be obtained without lowering the quantum yield; chemiluminescent life is still longer in a low chemiluminescent intensity system; the solubility to desired hydrophilic organic solvents is extremely high; and so forth. For instance, there has been offered DNPO as a chemiluminescent reagent with extremely excellent sensitivity [Rauhut et al., J. Amer. Chem. Soc., 89, 6515(1967)], but it was not satisfactory in terms of requirements other than the sensitivity, that is, this chemiluminescent substance has the defects that it does not have the stability required to be offered for the scientific world and the solubility to the hydrophilic organic solvents is low.